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Abstract

Hydrogen peroxide (H2O2) is discussed as being a signaling molecule in Arabidopsis (Arabidopsis thaliana) leaf senescence. Intracellular H2O2 levels are controlled by the H2O2-scavenging enzyme catalase in concert with other scavenging and producing systems. Catalases are encoded by a small gene family, and the expression of all three Arabidopsis catalase genes is regulated in a senescence-associated manner. CATALASE2 (CAT2) expression is down-regulated during bolting time at the onset of leaf senescence and appears to be involved in the elevation of the H2O2 level at this time point. To understand the role of CAT2 in senescence regulation in more detail, we used CAT2 promoter fragments in a yeast one-hybrid screen to isolate upstream regulatory factors. Among others, we could identify G-Box Binding Factor1 (GBF1) as a DNA-binding protein of the CAT2 promoter. Transient overexpression of GBF1 together with a CAT2:β-glucuronidase construct in tobacco (Nicotiana benthamiana) plants and Arabidopsis protoplasts revealed a negative effect of GBF1 on CAT2 expression. In gbf1 mutant plants, the CAT2 decrease in expression and activity at bolting time and the increase in H2O2 could no longer be observed. Consequently, the onset of leaf senescence and the expression of senescence-associated genes were delayed in gbf1 plants, clearly indicating a regulatory function of GBF1 in leaf senescence, most likely via regulation of the intracellular H2O2 content.

Plants have developed many different antioxidant enzymes and other compounds to detoxify ROS. Catalase (CAT; EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) are the key enzymes detoxifying hydrogen peroxide (H2O2; Asada, 1997; Willekens et al., 1997; Foyer and Noctor, 2000, 2009). Due to a very high apparent Km, catalases are not easily saturated with substrate and can act over a wide range of H2O2 concentrations, maintaining a controlled intracellular H2O2 concentration. Catalases provide the cell with a very energy-efficient mechanism to remove H2O2 because they decompose H2O2 without consuming cellular reducing equivalents. Even though plant catalases are predominantly located in the peroxisomes scavenging H2O2 produced by photorespiration (Scandalios et al., 1997; Foyer and Noctor, 2000), catalase action appears to be critical for maintaining redox balance during oxidative stress and is indispensable for stress defense in some C3 plants (Willekens et al., 1997).

In Arabidopsis, a small gene family has been characterized encoding catalases: CAT1, CAT2, and CAT3. All three Arabidopsis catalases show senescence-specific changes in expression and activity. CAT1 mRNA and CAT1 activity can only be detected in seeds and very late stages of leaf and plant senescence and are believed to play a role in detoxifying H2O2 produced during fatty acid degradation in glyoxysomes. CAT3 is expressed preferentially in the vascular bundles, and CAT3 expression and CAT3 activity can be induced by oxidative stress (Orendi et al., 2001; Zimmermann et al., 2006). CAT2 is predominantly expressed in photosynthetically active cells and is believed to mainly detoxify H2O2 produced during photorespiration (Zimmermann et al., 2006). Whereas CAT3 expression and CAT3 activity first increase during development and reach the maximum in 9-week-old plants, CAT2 expression and CAT2 activity decline when plants start to bolt (Zimmermann et al., 2006). In addition to the activity loss of CAT2 in photosynthetically active cells, APX1 activity also declines in leaf tissue during the transition from vegetative to reproductive growth, which coincides with the onset of leaf senescence. This leads to a reduced H2O2-scavenging capacity at this time point (Ye et al., 2000; Zimmermann et al., 2006). However, the APX1 mRNA level does not decrease in parallel (Panchuk et al., 2005); therefore, APX1 activity appears to be down-regulated on the posttranscriptional level. Remarkably, APX1 is rendered sensitive against its own substrate by a so far unknown mechanism in this developmental stage (Zimmermann et al., 2006). Hence, down-regulation of APX1 activity might be a consequence of down-regulation of CAT2 activity and increasing levels of H2O2. This suggests a feedback amplification loop in which CAT2 down-regulation would be the initial step. This coordinated regulation of the H2O2-scavenging enzymes on the transcriptional and posttranscriptional levels creates a distinct increase of H2O2 at the time point when the plants start to bolt, and a coordinated senescence process of all rosette leaves should be induced. Conversely, it was already shown that the senescence-regulating transcription factor WRKY53 and its regulators (Zentgraf et al., 2010) as well as other senescence-associated transcription factors (Balazadeh et al., 2008) and SAGs (Navabpour et al., 2003) can be induced by H2O2, so that the H2O2 peak during bolting time most likely serves as a signal to induce senescence-associated gene transcription. Since transcriptional down-regulation of CAT2 appears to be the initial step to create this senescence-promoting signal, we attempted to find factors influencing the transcription of CAT2 using CAT2 promoter fragments in a yeast-one-hybrid screen. We could identify G-Box Binding Factor1 (GBF1) as a CAT2 promoter-binding protein. Transient expression of 35S:GBF1 together with CAT2:GUS in tobacco (Nicotiana benthamiana) plants or Arabidopsis protoplasts revealed a negative effect of GBF1 on CAT2 expression. In gbf1 plants, the CAT2 decrease in expression and activity and the created H2O2 peak disappeared. Consequently, gbf1 plants showed a delayed senescence phenotype and an affected expression of SAGs.

RESULTS

GBF1 Binds to the CAT2 Promoter Both in Vitro and in Vivo

To identify proteins that are able to bind to the CAT2 promoter, a yeast one-hybrid screen was performed. Promoter CAT2:GUS deletion analyses revealed that the deletion of a 500-bp fragment approximately 850 bp upstream of the start codon led to the loss of CAT2 expression, indicating that the most important regulatory cis-elements are located in this region of the promoter (A. Smykowski, G. Orendi, and U. Zentgraf, unpublished data). Therefore, two overlapping CAT2 promoter fragments of this region were selected for the yeast one-hybrid screen (P1 and P2; Fig. 1A). A cDNA expression library of 6-week-old plants has been constructed and cloned into a vector containing the yeast GAL4 activation domain. This library was then screened by mating with a yeast strain containing a CAT2:His-3 construct and subsequent selection on His-free medium. Several candidate genes could be isolated. GBF1 (At4g36730) and two proteins with so far unknown functions (At4g29780 and At1g13930) appeared several times in the screen and were analyzed further. However, after retransformation of the full-length cDNAs of these candidate genes into yeast cells, only two proteins, GBF1 and the protein encoded by At4g29780, were able to induce yeast growth on selection medium, indicating CAT2 promoter binding (Fig. 1B). To confirm the binding of these proteins to the DNA fragments in vitro, a southwestern analysis was performed with extract of Escherichia coli BL21-SI cells expressing the full-length clones and the radioactively labeled promoter fragments P1 and P2. Therefore, the protein extracts were separated by SDS-PAGE, blotted on nylon filters, and incubated under DNA-binding conditions with radioactively labeled P1 or P2. The binding of only one of these three proteins, namely GBF1, with an expected protein mass of approximately 36 kD, could be confirmed (Fig. 1C; data not shown). The control E. coli BL21-SI cells with a transformed empty vector did not show a binding reaction (Fig. 1C). Western blot and immunodetection of the extract of E. coli BL21-SI cells expressing the 6×His-tagged GBF1 using anti-His antibodies revealed that the 36-kD protein on the southwestern blot is identical in size to the 6×His-tagged GBF1 (Fig. 1D). Obviously, GBF1 was able to bind to both CAT2 promoter fragments in the yeast system and in vitro. This is in agreement with the presence of a G-box, the DNA-binding motif of GBFs, in the overlapping region of P1 and P2 (Fig. 1A). An ELISA-based DNA-binding assay was established to analyze the DNA-protein interaction of GBF1 and the G-box in this CAT2 promoter region. Biotinylated P2 fragments containing either the G-box or a mutated G-box motif, in which the core sequence was exchanged to six adenines, were attached to streptavidin-coated ELISA wells. These plates were incubated with crude bacterial protein extracts with or without recombinant 6×His-tagged GBF1. After extensive washing, DNA-bound GBF1 was detected using an anti-His antibody conjugated with horseradish peroxidase and the substrate 1,2-phenylenediamine hydrochloride (Fig. 1E). Whereas the G-box-containing fragment was able to bind recombinant GBF1 in a concentration-dependent manner, the mutated version was not, clearly indicating that the G-box motif mediates the binding of GBF1 to the CAT2 promoter.

A, Schematic drawing of the CAT2 promoter region and the promoter fragments P1 and P2. B, Yeast one-hybrid analyses after mating of two yeast strains carrying full-length cDNA of GBF1 or two proteins of unknown function, At4g29780 and At1g13930 and P1:His-3 and P2:His-3 constructs, respectively, on selective or nonselective medium. C, Southwestern analyses of E. coli extracts expressing GBF1 using both promoter fragments, P1 and P2, of the CAT2 promoter. Extracts of an E. coli strain BL21-SI carrying an empty vector was taken as a control. D, Detection of recombinant 6×His-tagged GBF1 by western blot and immunodetection using anti-His antibodies. Extracts of E. coli strain BL21-SI carrying an empty vector were used as a negative control. E, ELISA-based DNA-protein interaction assay using different dilutions of crude E. coli extracts with and without recombinant 6×His-tagged GBF1 and biotinylated P2 promoter fragments containing a G-box (black bars) or a mutated G-box (G-mut; white bars). F, Activation of the Gal1:LacZ reporter gene construct by full-length GBF1 fused to the yeast GAL4-binding domain. The empty vector was taken as a control. Activation was examined through X-Gal overlay assay after 5.5 h (left) and 23.5 h (right). Expression of the recombinant proteins (GBF1-GDB and GBF1Δ95-GDB) was confirmed by western blot.

GBF1 Activation Potential

To determine whether GBF1 can act as a transcriptional activator, the full-length cDNA sequence fused to the yeast GAL4 DNA-binding domain was used as an effector construct in the yeast one-hybrid system. The expression of the LacZ reporter gene under the control of the GAL1 promoter was visualized by an X-Gal agarose overlay assay (Fig. 1F). The yeast strain transformed with the cDNA coding for GBF1 was able to activate the reporter gene expression, whereas a GBF1 deletion construct (GBF1Δ95) missing 95 amino acids of the N terminus and the empty vector showed no detectable expression. This indicates that GBF1 contains a functional transcriptional activation domain in the N-terminal region. The expression of the recombinant proteins in the yeast cells was confirmed by western blot and immunodetection (Fig. 1F).

GBF1 Negatively Regulates CAT2 Expression

The in vivo effect of GBF1 on CAT2 expression was investigated in two different transient expression systems using CAT2:GUS as a reporter. Leaves of tobacco were coinfiltrated with Agrobacterium tumefaciens containing the CAT2:GUS reporter construct and A. tumefaciens containing either a 35S:GBF1 expression vector or an empty vector. GUS activity of 16 independently transformed plants was measured. The GUS activity determined in plants transformed with the reporter construct and the empty control vector was defined as basal GUS activity and was used as a reference and set to 100%. Cotransformation of the reporter construct with a 35S:GBF1 expression vector reduced the basal GUS activity about 35% (Fig. 2A). In addition, Arabidopsis protoplasts were transformed with the respective plasmids. Double transformations with a CAT2:GUS construct and either a 35S:GBF1 construct or an empty vector were conducted. The basal GUS activity was determined after transformation of the CAT2:GUS construct and an empty 35S expression vector and was set to 100%. Again, transformation of a CAT2:GUS construct together with a 35S:GBF1 expression vector reduced the activity of GUS significantly compared with the basal activity (Fig. 2B). Thus, we assume that GBF1 works as a repressor for CAT2 expression even though GBF1 has a functional activation domain. However, the G-box is located more than 1,000 bp upstream of the transcriptional start site, so that the activation domain might not directly interact with the basal transcription machinery.

A, Tobacco leaves were coinfiltrated with A. tumefaciens transformed with a CAT2:GUS construct and A. tumefaciens containing either an empty 35S plant expression vector as a control or a 35S:GBF1 expression vector. GUS activity levels in control transformations were set to 100%. Sixteen independent experiments were performed. The error bars indicate sd (t test, P = 0.0217). B, Arabidopsis protoplasts were cotransformed with a CAT2:GUS reporter construct and either an empty 35S plant expression vector as a control or a 35S:GBF1 expression vector. GUS activity levels in control transformations were set to 100%. Nine independent transformation experiments were performed. Error bars indicate sd (t test, P = 0.006545).

To verify the effect of GBF1 on CAT2 expression in planta, we characterized two different GBF1 T-DNA insertion lines. One T-DNA insertion was localized in exon 9 (gbf1-ex; SALK_144534) and the other one in intron 4 (gbf1-int; SALK_147518) of the GBF1 gene. Plants that were homozygous for these insertions were characterized by a PCR screen. The GBF1 expression was analyzed in comparison with ecotype Columbia (Col-0) wild-type plants, which revealed a more or less constant expression of GBF1 in 4-, 6-, and 8-week-old plants. Whereas in gbf1-ex plants no expression could be detected, the insertion of the T-DNA in the intron in gbf1-int plants led to a reduced expression of GBF1 (Fig. 3A). Genevestigator data (http:/www.genevestigator.ch) and GBF1:GUS plants confirmed a constant expression level of GBF1 throughout leaf and plant development (Supplemental Fig. S2). However, if GBF1 negatively regulates CAT2 expression, CAT2 mRNA levels should be altered in gbf1-ex and gbf1-int. Quantification of reverse transcription (RT)-PCR analyses revealed that the down-regulation of CAT2 expression with increasing plant age in Col-0 plants disappeared in gbf1-ex and gbf1-int (Fig. 3B). Protein levels of the different catalases were determined by native PAGE of plant protein extracts and subsequent western blot with an immunodetection using anti-rye (Secale cereale) catalase antibodies. In agreement with gene expression, the protein level of CAT2 decreased with plant age and the progression of senescence in wild-type plants. In contrast, CAT2 protein content remained constant in gbf1-int extracts or even increased in gbf1-ex plant extracts. CAT3 mRNA and protein levels increased in all three plant extracts, but to a lesser extent in gbf1-int and gbf1-ex plants (Figs. 3C and 4A). CAT1 mRNA and protein could not be detected in these developmental stages (Figs. 3D and 4A). The enzyme activity of the different enzyme isoforms was determined by native PAGE followed by a catalase-specific activity staining. As shown before, CAT2 activity decreased in parallel with mRNA and protein levels in wild-type plants with progression of senescence (Zimmermann et al., 2006). In gbf1-ex plants, no decrease in CAT2 activity could be observed, whereas in gbf1-int plants, CAT2 activity decreased but to a lesser extent compared with the wild type (Fig. 4B). With the loss of CAT2 down-regulation in gbf1-ex plants, the H2O2 peak, which can be observed in wild-type plants during bolting, also disappeared (Fig. 5; Miao et al., 2004; Zimmermann et al., 2006). Taken together these results clearly indicate that GBF1 is involved in the senescence-associated down-regulation of CAT2 expression, resulting in an activity loss of CAT2 and an increase in H2O2 levels. Senescence-associated CAT3 induction appears to be slightly affected in gbf1-ex and gbf1-int compared with the wild type, which correlates with the delayed senescence phenotype of gbf1-ex and gbf1-int plants described below. To corroborate the results of the knockout plants, we constructed 35S:GFB1-overexpressing plants. After transformation, we received several Basta-resistant transgenic lines; however, the overexpression of GBF1 always led to silencing of the endogenous GBF1 gene and the transgene (Supplemental Fig. S1). In agreement, the phenotype of these transgenic lines was similar to gbf1-int.

Expression analyses of GBF1 (A), CAT2 (B), CAT3 (C), and CAT1 (D) in Col-0 wild-type and gbf1-ex and gbf1-int plants using RT-PCR. ACTIN2 was used as a control for equal amounts of cDNA. One example of an agarose gel and the quantification of expression analyses of three independent experiments are presented. Error bars indicate sd.

A, Western-blot analyses of a native protein gel of 4-, 6-, and 8-week-old Col-0 and gbf1 plant extracts using anti-rye catalase antibodies for immunodetection. B, Catalase zymogram of 4-, 6-, and 8-week-old Col-0 and gbf1-ex and gbf1-int plant extracts. Leaves 5, 6, 7, and 8 of approximately five plants were pooled for each experiment. The experiment was repeated five times, always showing the same activity pattern; one representative example is shown here. Activity was quantified using the NIH Image program and expressed as relative intensity (Rel. Int.) in percent, referring to the value in 4-week-old plants.

Relative H2O2 concentration was measured per leaf in 5- to 10-week-old Col-0 and gbf1-ex plants. The values in 4-week-old Col-0, gbf1-int, and gbf1-ex plants were set as reference to 100%. Error bars indicate the sd of three to five independent experiments.

Phenotype- and Senescence-Associated Gene Expression

The phenotypes of gbf1-ex and gbf1-int plants were analyzed in comparison with Col-0 wild-type plants. No difference in overall development, bolting, and flowering time could be observed (Fig. 6B). However, if rosettes of 6.5-week-old plants were analyzed upside down, wild-type plants already showed several yellow leaves whereas gbf1-ex and gbf1-int plants did not. Using a specific color code, leaves were sorted according to their age. The old leaves of the wild-type plants had already turned yellow or brownish, while the leaves of gbf1-ex and gbf1-int plants were still green even though chlorophyll loss had already started and become visible (Fig. 6A). In 8.5-week-old plants, gbf1-ex and gbf1-int plants still had fewer yellow and brownish dry leaves than wild-type plants. This indicates that gbf1-ex and gbf1-int plants show a delay in leaf senescence that is not coupled to a delay in general development. To verify delayed senescence in the mutant lines, we analyzed SAG12 and WRKY53 as two examples for early and late senescence-induced genes and RBCS1a as one example for a senescence-down-regulated gene. In accordance with the phenotype, expression of SAG12 and WRKY53 was not induced or only to a lower extent in gbf1-ex and gbf1-int plants of the same age and developmental stage (Fig. 7). In contrast, expression of the senescence regulator WRKY53 appears to be repressed in the mutants. This effect appears to be more pronounced in the knockout mutant gbf1-ex than in the knockdown mutant gbf1-int. This repression appears to be released again in gbf1-int in 8-week-old plants. As expected, RBCS1a is down-regulated in the wild-type plants with increasing age. This down-regulation is diminished in the mutant lines, indicating that senescence is delayed. In addition, this resembles very much the loss of CAT2 down-regulation in the mutants, also supporting a direct negative regulation of RBCS1a by GBF1. In agreement, the RBSC1a promoter contains a G-box, and we could show that GBF1 directly interacts with this G-box in an ELISA-based DNA-binding assay (Supplemental Fig. S3). bZIP63, belonging to the C-group bZIP factors and binding the C-box, was used as a negative control and did not bind to either the CAT2 or the RBCS1a G-box fragment (data not shown). GBF1 was already identified as a negative regulator of RBCS1a in tomato (Solanum lycopersicum) plants and in Arabidopsis (Giuliano et al., 1988; Mallappa et al., 2006).

Phenotypes of Col-0 and gbf1 plants. A, Rosette leaves of 6.5-week-old plants and 8.5-week old plants sorted from young to old leaves within the rosette using a color code. Upside-down rosettes are shown on the right side. B, Development of plants from 4 to 7 weeks.

Expression of GBF1, WRKY53, RBCS1a, and SAG12 in 4-, 6-, and 8-week-old Col-0, gbf1-ex, and gbf1-int was examined by semiquantitative RT-PCR. ACTIN2 was used as a control for equal amounts of cDNA. One example of an agarose gel (A) and the quantification of expression analyses of three independent experiments (B) are presented. Error bars indicate sd of five independent experiments.

DISCUSSION

Despite the enormous agricultural importance of senescence processes, we are far from understanding the complex regulatory network, with all its convergence nodes, that governs senescence. There are many indications that H2O2 acts as a signal molecule to induce senescence. Important senescence-associated transcription factors of Arabidopsis like WRKY53 and its regulators are transcriptionally up-regulated by increasing levels of H2O2 (Miao et al., 2004, 2007, 2008). Consistently, the H2O2 concentration increases during bolting in Arabidopsis, which coincides with the induction of senescence in the whole rosette. This increase appears to be initiated by a transcriptional down-regulation of CAT2 (Zimmermann et al., 2006). Therefore, we analyzed the CAT2 promoter region to isolate interacting proteins using the yeast one-hybrid system. By screening a cDNA library of 6-week-old leaf material, we found three candidates that appeared several times in the screen, the transcription factor GBF1 and two proteins with so far unknown function. We concentrated our further analyses on GBF1, since southwestern blotting could confirm only the binding of GBF1 to P1 and P2 promoter fragments. GBF1 belongs to the family of bZIP transcription factors, which has at least 75 representatives regulating diverse biological processes such as pathogen defense, light and stress signaling, seed maturation, and flower development. This transcription factor family was divided into 10 groups of bZIP proteins based on sequence similarity of the basic region and the presence of additional conserved motifs (Jakoby et al., 2002). GBF1 was assigned to the G group. The group G bZIPs of Arabidopsis and their parsley (Petroselinum crispum) homologues were mainly related to UV and blue light signaling (Weisshaar et al., 1991; Schindler et al., 1992b; Kircher et al., 1998). In vitro analyses have shown that the GBF proteins can bind as homodimers and heterodimers to symmetric and asymmetric G-boxes present in light-responsive promoters (Armstrong et al., 1992; Schindler et al., 1992b). Here, we could show that GBF1 binds to two overlapping DNA fragments of the CAT2 promoter containing a symmetric 5′-CACGTG-3′ G-box motif in the overlapping sequences, suggesting a role of GBF1 in the regulation of CAT2 expression and H2O2 levels.

Even though we could confirm that the N-terminal Pro-rich region can function as a transcription activation domain, transient overexpression of GBF1 together with a CAT2:GUS construct in tobacco leaves or Arabidopsis protoplasts revealed that GBF1 expression led to a decline in GUS expression, indicating that GBF1 acts as a negative regulator for CAT2 expression. However, if GBF1 acts as negative regulator responsible for the down-regulation of the CAT2 gene during bolting and the onset of senescence, and if this down-regulation of CAT2 really is the initial step to create the H2O2 peak at this time point, a gbf1 mutant plant should not exhibit this CAT2 down-regulation. Accordingly, the H2O2 peak should be gone and senescence should be delayed. This is exactly what was observed in two T-DNA insertion lines of GBF1. In the knockout line gfb1-ex as well as in the knockdown line gbf1-int, CAT2 expression as well as CAT2 protein content and activity did not decrease, at least not to the same extent as observed in wild-type plants. The H2O2 peak, which was observed in wild-type plants, disappeared in gbf1 mutant plants. In agreement, expression of the senescence-associated and H2O2-induced transcription factor WRKY53, as well as the senescence marker gene SAG12, was diminished. Senescence-associated down-regulation of RBCS1a expression was abolished and senescence of the rosette leaves was delayed. In contrast to Mallappa et al. (2006), we could not observe an early-flowering phenotype in our gbf1 plants, which might be due to different light conditions, even though the gbf1 mutants sometimes had fewer rosette leaves when the plants started to bolt. Taken together, this clearly indicates that GBF1 is involved in the regulation of the onset of senescence, most likely through the regulation of CAT2 expression and intracellular H2O2 content. This is in accordance with studies on other mutants exhibiting a senescence phenotype; for example, timing of senescence is altered in vtc-1, which is deficient in ascorbate production (Barth et al., 2004), and in onset of leaf senescence5, which is disrupted in quinolinate synthase, an enzyme involved in de novo NAD synthesis (Schippers et al., 2008), and in ore9, which is more tolerant of oxidative stress and encodes an F-box protein involved in protein degradation (Woo et al., 2004). In general, ROS, antioxidants, and redox state undergo significant changes with leaf age and are thought to be involved in senescence regulation (Zentgraf and Hemleben, 2008; Foyer and Noctor, 2009), but senescence regulation is a very complex process integrating many different endogenous and exogenous signals. Therefore, oxidative stress in CAT2 knockout mutants does not simply induce premature senescence (Queval et al., 2007).

However, GBF1 itself appears not to be regulated predominantly on the transcriptional level. Our RT-PCR data, GBF1:GUS plants, and also Genevestigator and Arabidopsis eFP Browser data revealed that GBF1 is only expressed at low to moderate levels, as expected for a transcription factor, and is present in many different tissues. Its expression appears to be more or less constant over leaf development, with a slight up-regulation during bolting and in senescent leaves. Expression can be slightly induced by osmotic or drought stress and is only slightly modulated by hormone treatments. However, it has been known for many years that GBFs are extensively regulated on the posttranscriptional level. DNA-binding activity of the Arabidopsis GBF1 is stimulated by phosphorylation through casein kinase II (Klimczak et al., 1992, 1995). Furthermore, GBF1, GBF2, and GBF3 can also form heterodimers, suggesting a potential mechanism for generating additional diversity in regulation mechanisms by these GBF proteins (Schindler et al., 1992a). Recently, it was shown that AtbZIP16 and AtbZIP68, also belonging to the G group, could form heterodimers with the other members of the G group (Shen et al., 2008).

In parsley, a light-modulated transport of GBFs to the nucleus could be observed (Harter et al., 1994). Arabidopsis GUS:GBF1 fusion proteins localized 50% to 62% in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 fusions were localized in the nucleus. By contrast, about 50% of GUS:GBF2 was found in the cytoplasm of dark-grown cells, whereas over 80% of this protein was found in the nucleus in cells cultured under blue light. Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention. These results suggest that limitation of nuclear access may also be an important control of GBF activity (Terzaghi et al., 1997). In addition, yeast two-hybrid assays and in vitro binding assays indicated that the GARP transcriptional activator GPRI1 (for GBF's Pro-rich region-interacting factor 1) can interact with the Pro-rich regions of GBF1 and GBF3, whereas GPRI2 interacted only with the Pro-rich region of GBF1 (Tamai et al., 2002). GPRI1 and GPRI2 may function in concert with a GBF1 through interaction with its Pro-rich region to modulate the transcriptional level of specific target genes. Moreover, interaction with another protein, GBF-Interacting Protein1 (GIP1), resulted in a 10-fold increase in GBF1 DNA-binding activity without altering the migration in an electrophoretic mobility shift assay. This suggests a transient association of GIP1 and GBFs and a possible action as a potent nuclear chaperone or crowbar, potentially regulating the multimeric state of GBFs (Sehnke et al., 2005). Recently, it was shown that GBF1 is degraded by a proteasome-mediated pathway independent of COP1 and SPA1. Furthermore, COP1 physically interacts with GBF1 and is required for the optimum accumulation of GBF1 protein in light-grown seedlings (Mallappa et al., 2008).

How GBF1 activity is regulated in a developmental and senescence-associated manner still has to be elucidated. In vivo localization and in vivo interaction studies with the above-mentioned candidates will be carried out to learn more about the mechanism of senescence-specific GBF1 regulation.

MATERIALS AND METHODS

Plant Material

Seeds from Arabidopsis (Arabidopsis thaliana Col-0) were grown for 16 h in an illuminated climate chamber at 22°C (60 μmol s−1 m−2). Under these conditions, the plants developed flowers within 7 weeks and the mature seeds were harvested after 12 weeks. During growth and development of the leaves, the respective positions within the rosette were color coded with different colored threads, so that even in very late stages of development individual leaves could be analyzed according to their age (Hinderhofer and Zentgraf, 2001). Leaves of the same position within different rosettes were pooled. Since CAT2 and CAT3 are under circadian regulation, leaves were always harvested 3 h after the beginning of illumination. Leaves were pooled in all experiments; series of experiments were repeated three to eight times.

Protein Extraction, Native PAGE, and Catalase Activity Staining

Whole leaves were homogenized in 100 mm Tris-HCl, pH 8.0, 20% glycerol, and 30 mm dithiothreitol on ice and centrifuged at 14,000g for 30 min at 4°C. Total protein content was quantified according to the method of Bradford (1976) and directly used for native PAGE. Five micrograms of protein extract was separated on a 6% polyacrylamide gel (0.12 m Tris-HCl, pH 6.8, as gel buffer) without stacking gel in the absence of SDS. Subsequently, the gels were incubated in 0.01% H2O2 solution for 2 min and washed twice in water. Afterward, the gels were soaked in a 1% solution (w/v) of FeCl3 and K3[Fe(CN)6] for 4 min and washed vigorously (Zimmermann et al., 2006). Activity was quantified using the NIH Image program and expressed as relative intensity in percent, referring to the value in 4-week-old plants.

Western-Blot Analysis of Catalases

Twenty micrograms of protein extract was separated on a 6% native PAGE gel and transferred onto a nitrocellulose membrane by semidry blotting. The membrane was washed twice with Tris-buffered saline (TBS) and then blocked with 3% milk powder in TBS. The detection was performed with polyclonal anti-rye (Secale cereale) catalase antibodies in 1.5% milk powder. After washing twice with TBS-Tween 20 (TBS-T), the secondary peroxidase-conjugated antibodies were used for the chemiluminescence detection of catalases.

Recombinant Proteins

The full-length GBF1 cDNA fragment was cloned into the pQE30 expression vector (Qiagen) to create pQE-GBF1 encoding the 6×His-tagged protein. Escherichia coli cells carrying the plasmid were cultured in Luria-Bertani broth and treated with 1 mm isopropylthio-β-galactoside for induction of the recombinant protein syntheses. The harvested cells were disrupted by sonication and centrifuged. Clear lysates were used for western and southwestern analyses.

Western-Blot Analysis of His-Tagged GBF1

Proteins were separated on 12% polyacrylamide gels and transferred to nitrocellulose membranes by semidry blotting. Membranes were blocked for 1 h at room temperature in TBS-T containing 5% (w/v) nonfat dry milk powder. The membranes were incubated with anti-His antibodies for 1 h. Blots were washed in TBS-T for 10 min (three times) before incubation with secondary antibodies conjugated with a peroxidase. The blots were washed with TBS-T for 10 min (three times), and antibody conjugates were detected by chemiluminescence and exposure to x-ray films.

Southwestern Analyses

Proteins were loaded on a SDS-PAGE gel, subjected to electrophoresis, and transferred to a nitrocellulose membrane. The nitrocellulose membrane was incubated with 1× binding buffer containing 10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 20% glycerin, and 200 μg of heat-denatured, ultrasound-treated calf thymus DNA for 1 h at room temperature. Subsequently, 500 ng of radioactively labeled CAT2 promoter fragments (P1 and P2) was added and incubated for another 1 h at room temperature. Then, the blot was washed three times for 20 min at room temperature with 1× binding buffer, air dried, and exposed to x-ray film. The purified PCR fragments were end labeled using [γ-32P]dATP and T4 polynucleotide kinase.

ELISA-Based DNA-Binding Assay

A total of 100 mL of culture of E. coli cells (BL21) expressing 6×His-tagged GBF1 was lysed in 1 mL of lysis buffer (50 mm Tris, pH 7.5, 50 mm NaCl, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, and 0.02% Nonidet P-40). Using this bacterial protein extract, the DNA-binding assay was performed as described by Kirchler et al. (2010). The biotinylated PCR product (P2; 190 bp) containing the wild-type or mutated G-box or a RBCS1a promoter fragment also containing a G-box (5′-AATTATCTTCCACGTCGATTATTCC-3′) or a mutated version was attached to streptavidin-coated ELISA wells (Nunc Immobilizer) and blocked with 1% blocking agent (Roche). Crude protein extracts were added to each well in different dilutions (1:50, 1:100, 1:200), incubated for 1 h, and washed with blocking buffer (Qiagen) containing 0.1% Tween 20. Anti-His antibody conjugates (Penta-His HRP; Qiagen) were added to each well followed by 1 h of incubation at room temperature. After washing with blocking buffer (Qiagen), the substrate 1,2-phenylenediamine hydrochloride (Dako) was added to the wells. The reaction for the detection of DNA-bound protein was performed according to the manufacturer's instruction (Dako). The A492 was determined using an ELISA plate reader (Tecan) with filter setting at 650 nm for reference.

Construction of the CAT2 Promoter-GUS Fusion and Transient Expression Assays

The 1.4-kb promoter region upstream of the CAT2 coding region was amplified (forward primer, 5′-AATTCGGCAATTCAGTACCATG-3′, reverse primer, 5′-GGTTGATGAGAAGAGAGCTTGG-3′) from Arabidopsis DNA and cloned upstream of the GUS reporter gene of the PCB308 binary vector (Xiang et al., 1999). The coding sequence of GBF1 has been cloned adjacent to a cauliflower mosaic virus 35S promoter of the PY01 binary vector. Transient expression analyses were performed using the isolated plasmids and Arabidopsis mesophyll protoplasts as described by Sheen (2001). GUS activity assay was carried out as described by Jefferson et al. (1987). For transient expression in tobacco (Nicotiana benthamiana), leaves were infiltrated with Agrobacterium tumefaciens (strain GV3101) that had been transformed with the CAT2:GUS construct, the 35S:GBF1 construct, or the empty 35S vector as a control. Furthermore, A. tumefaciens containing P19 for inhibition of silencing and DsRed for normalization, both of which were cloned into the binary vector BinAR, was added. All bacterial suspensions were diluted to an optical density of 0.8, mixed, and then applied to the bottom side of the tobacco leaves using a needleless syringe. One-half of a leaf was transformed with the CAT2:GUS and the 35S:GBF1 constructs, whereas the other half was transformed with the CAT2:GUS construct and the empty 35S vector as a control. After 48 h, leaf discs (approximately 1 cm2) were punched out of the transformed leaf areas, and the GUS activity assay was carried out as described by Jefferson et al. (1987).

Construction of GBF1 Promoter-GUS and 35S:GBF1-Overexpressing Lines

The area 1.5 kb upstream of the start codon was amplified from genomic DNA and cloned into the vector pCB308 in front of the GUS gene via BamHI and XbaI. The GBF1 coding sequence was cloned into the vector PY01 adjacent to a cauliflower mosaic virus 35S promoter. Both constructs were verified by sequencing. Arabidopsis transformation was performed by the vacuum infiltration procedure (Bechtold and Pelletier, 1998). The seeds of the transgenic plants were selected by spraying with 0.1% Basta.

Semiquantitative RT-PCR

Total RNA was isolated from whole leaves using the Purescript RNA Isolation Kit (Gentra, Biozym) and subsequently reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad) as recommended by the manufacturer. PCR on cDNA was performed with CAT2 primers (forward, 5′-CAGGTTCGTCATGCTGAGAAG-3′; reverse, 5′-TTAGATGCTTGGTCTCACGTT-3′), CAT3 primers (forward, 5′-CAAACAGGCTGGAGACAGGT-3′; reverse, 5′-GACGGATTTAACGACCAAGC-3′), CAT1 primers (forward, 5′-TCATCGGGAAGGAGAACAAC-3′; reverse, 5′-ACCAAACCGTAAGAGGAGCA-3′), GBF1 primers (forward, 5′-GGTCGAAAGATGGTGAAGGA-3′; reverse, 5′-ATCCGATTCCAATCACGAAG-3′), RBCS1a primers (forward, 5′-ATTGCCTACAAGCCACCAAG-3′; reverse, 5′-ATTTGTAGCCGCATTGTCCT-3′), WRKY53 primers (forward, 5′-GATCACAAGAACACCACCATTAGCC-3′; reverse, 5′-AAAGTTGTGTCAATCTCGACCGTTG-3′), as well as SAG12 primers (forward, 5′-CCCGGTTAATGATGAGCAAGC-3′; reverse,5′-GCTTTCATGGCAAGACCACA-3′). RT-PCR products were separated on 1% agarose gels. The intensity of the bands was determined using the NIH Image program Scion Image (Scion Corporation). The mRNA levels were normalized with respect to the level of mRNA for ACTIN2, which was taken as a reference (Panchuk et al., 2005).

Yeast One-Hybrid Analyses

The Matchmaker System from BD Biosciences Clontech was used to perform the yeast one-hybrid screen. CAT2 promoter fragments (fragment P1, position −1,050 upstream of ATG, length 172 bp, forward primer 5′-AGATGAACTTCTGAGCGAGC-3′, reverse primer 5′-GGATTCCACGTGTGATGAG-3′; fragment P2, position −900 upstream of ATG, length 190 bp, forward primer 5′-CACTATCTCATCACACGTGG-3′, reverse primer 5′-AGCAAGGAAGAAGGACGACA-3′) were cloned upstream of the reporter gene in the vector pHISi, linearized, and integrated into the yeast genome of Y187. A second yeast strain (AH109) was then transformed with a library of cDNAs obtained from 6-week-old leaf material that had been fused to the activation domain of the reporter gene by cloning into the vector pGADT7Rec. Mating of the two strains was conducted at 30°C overnight. To reduce false positives, a small amount of 3-amino-1,2,3-triazole was added. The one-hybrid screenings and assays were performed as described in the Matchmaker One-Hybrid System protocol (BD Biosciences Clontech). Full-length cDNAs were isolated for the candidate genes that had been isolated several times in the screen, cloned into the pGADT7Rec vector, and transformed into Y187 containing the CAT2 promoter reporter construct.

To analyze the activation potential of the GBF1 protein, the full-length cDNA was inserted into pGBKT7 vector to generate a GBF1-GBD (GAL4 DNA-binding domain) fusion construct. These plasmids were introduced into yeast strain Y187 containing the LacZ gene under the control of the GAL1 promoter. β-Galactosidase activity measurement and X-Gal agarose overlay assay of yeast were performed according to the protocol of the Herskowitz laboratory using o-nitrophenyl β-galactopyranoside as a substrate (http://biochemistry.ucsf.edu/∼herskowitz/protocols.html).

H2O2 Measurement

H2O2 was determined by incubating one Arabidopsis leaf in 1 mL of 10 μm 2′7′-dichlorohydrofluorescein diacetate (Invitrogen) for 45 min at room temperature. Plant tissue was rinsed in water, frozen in liquid nitrogen, and homogenized with a mortar after addition of 1 mL of 40 mm Tris-HCI buffer, pH 7.0. The homogenate was centrifuged at 20,600g for 15 min, and the supernatant was recovered. Fluorescence of the clear extract was determined in a spectrofluorometer (Berthold) using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Overexpression of GBF1 under the control of the 35S promoter led to gene silencing of the transgene and the endogenous gene copy.

Supplemental Figure S3. Analyses of a direct interaction of GBF1 with the G-box of the RBCS1a promoter.

Acknowledgments

We thank Prof. Dr. J. Feierabend (University of Frankfurt) for providing the anti-rye catalase antibodies and the Nottingham Arabidopsis Stock Centre for the donation of seeds for the T-DNA insertion lines.

Footnotes

↵1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. SFB446).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ulrike Zentgraf (ulrike.zentgraf{at}zmbp.uni-tuebingen.de).

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